The initial Framingham epidemiological study established an indisputable link between lipids and coronary artery disease (CAD), and since the time of that initial study medical research efforts have advanced the understanding of lipid metabolism as well as the mechanisms of atherogenic processes which underlie CAD. There has been a corresponding effort to develop appropriate diagnostic tests which can identify individuals at risk for the disease and detect the disease in an early stage in order to provide effective preventive diet and drug therapy. All lipoprotein classes are similar in that they have a hydrophobic core of non-polar lipids comprising triglycerides and cholesterol esters. They are coated, with phospholipids and cholesterol, in which specific proteins, the apolipoprotein, are embedded.
Lipoproteins are classified on the basis of their hydrated density. Accordingly, they are separated on ultracentrifugation into large, low density chylomicrons(&lt;1.006 gm/mL;&gt;100 nm), very low density lipoproteins (VLDL, d &lt;1.006 gm/mL; 30-90 nm), intermediate density lipoproteins (IDL, d=1.006-1.019 gm/mL), low density lipoproteins (LDL, d=1.025-1.063 gm/mL; about 20 nm), and high density lipoproteins (HDL, d=1.063-1.21 gm/mL; about 8-12 nm).
The lipoprotein classes can also be identified by means of their associated apolipoproteins. Fourteen major human plasma apolipoproteins have been identified and their associations with lipoproteins characterized. The two major apolipoproteins on HDL are apo A-I and apo A-II. Chylomicrons are associated with apo B.sub.48, and apo B.sub.100 is the predominate apolipoprotein on VLDL and LDL. The apo C proteins are associated with all lipoproteins except LDL. Apolipoprotein E is a constituent of chylomicrons, VLDL, and HDL. Other apolipoproteins, such as Lp(a), apo D and apo F are present in low concentrations. Their significance is poorly understood.
The function of the lipoproteins in lipid metabolism is primarily one of transport, but is nonetheless quite complex. Metabolism pathways comprise an exogenous system, wherein the lipoproteins transport triglycerides and cholesterol esters as well as other dietary lipids out of the intestine, and an endogenous system, wherein they transport corresponding hepatic lipids. The apolipoproteins on lipoproteins, by interacting with enzymes and cell surface receptors, direct lipoprotein lipids to an appropriate site of metabolism.
Since the defect in metabolism leading to atherosclerosis may be multifactorial, it is difficult to select the preferred marker or combination of markers to identify those patients who have an increased risk for premature cardiovascular disease. As the understanding of lipid metabolism has undergone rapid evolution, so have the clinical tests used to evaluate and predict CAD. Among the diagnostic ratios in present use are total cholesterol/HDL cholesterol, LDL cholesterol/HDL cholesterol, VLDL cholesterol/triglyceride, cholesterol/triglyceride, LDL cholesterol/Apo B, and HDL cholesterol/Apo A.sub.1. Currently Apo A.sub.1 /ApoB is considered the best predictive ratio, and Lp(a), a cholesterol rich lipoprotein with pre-.beta. mobility, the best independent risk marker.
One criticism of all these determinations is that they do not measure intact functional lipoproteins, but extracted or separated components. This may be particularly disadvantageous in the case of the apolipoprotein measurements. Studies show that immunological determinations of apolipoproteins vary with the conformation of the proteins, whether native or denatured, and also that their immunological reactivity is affected by lipid association, with greater numbers of epitopes being unmasked or expressed and available for antibody binding as lipid is lipolysed away. In the process of apolipoprotein isolation, lipoprotein lipid moiety is lost either inadvertently in the process of centrifugation, or purposefully, when lipases or detergents are used in separations, or preliminary to apolipoprotein quantitation. It is estimated that 35% of plasma Apo A.sub.1 is lost upon prolonged centrifugation (Kunitake et al., J. Lipid Research 23:936-40 (1982)). As a result, normal apolipoprotein values cover a broad range and individual determinations are poorly reproducible.
Certain current methodologies for quantification of cholesterol and triglycerides in human serum using either chemical or enzymatic methods use solvent extraction or enzymatic hydrolysis of the substrates, a step which destroys the normal "anatomy" of lipoprotein particles. Both triglyceride and cholesterol values are very crude indicators of lipid moiety from lipoprotein particles. For example, triglycerides are estimated in total serum, not in VLDL, LDL, and HDL fractions; the cholesterol is chemically quantitated only for total cholesterol and HDL cholesterol; and LDL cholesterol and VLDL cholesterol are only indirectly computed.
The estimation of HDL cholesterol in lipemic samples (particularly postprandial samples) is exposed to errors due to the inability to precipitate large particles of chylomicrons, VLDL and LDL of lower density with manganese and heparin. There are large interlaboratory variations for cholesterol as well as for triglycerides, and usually no correction is made for free cholesterol. Therefore, the cutting point at which to evaluate patients at risk is very imprecise and, as a result, they may be committed to longterm diet or treatment with drugs unnecessarily.
The chemical isolation of the lipid fraction is an artificial approach disrupting the normal anatomy of lipoprotein particles. Triglycerides and cholesterol in body fluids perform their function not as isolated lipid fractions, but incorporated and intermingled with each other in lipoprotein particles, the normal way these lipid fractions operate in normal conditions in the body fluids.
Until now we quantified cholesterol and triglycerides because these were the techniques available. Presently we are able to isolate apo B.sub.100 and apo A.sub.1 -containing particles by capturing them with specific antibodies. On these isolated particles I suggest now the study of the lipid moieties, a more meaningful approach.
The same considerations are true apolipoproteins which do not circulate in the blood as isolated molecules, but as a component of an intact lipoprotein particle. In fact, even the International Union of Immunological Societies (IUIS) recommended as a standard for apo A.sub.1 and apo B.sub.100, a lyophilized pool of serum which contains intact lipoprotein particles (apo A.sub.1 and apo B.sub.100 -containing particles (Naito, H., Clinical Chemistry 34(8):B.sub.84-94 (1988). Currently, both lipid fractions and apolipoproteins are expressed in mass units (mg/dl). The use of absolute mass units or absolute immunoreactive mass units is not essential. It is difficult to express a function or phenomenon, such as the masking or unmasking of apolipoproteins epitopes by lipids in fast or after meals, in mass units. What is essential in these studies is the direction of a phenomenon which can be detected using the relative units in multiple samples in a dynamic approach before and after meals, during a day.
The evaluation of lipoprotein particles by quantitating the amount of lipids only (triglyceride and cholesterol) was considered in the past to be misleading (Avogaro, P. et al., Lancet I: 901-3 (1979); Maciejko, J. J. et al., New Engl. J. Med. 309:385-389 (1983); and Sniderman et al. Proc. Natl. Acad. Sci. 77(1):604-608 (1980)). The apolipoprotein component of the lipoprotein was suggested as more stable, and hence more suitable for diagnostic purposes. This view of "stability" is contradicted by my invention because it ignores the fact that both lipid and apolipoprotein component are extremely variable due to their dynamic changes in a normal lipoprotein particle physiology. In fact, the very essence of their function implies continuous changes in lipid components or in lipid moiety concentrations and apolipoprotein epitope expression. Therefore, my approach, instead of rejecting or ignoring this important fact recognizes the variability of both components (lipid moiety and apolipoprotein) and develops a method of studying this variability of lipid and apolipoprotein components in a dynamic fashion in an intact lipoprotein particle.
Unlike currently performed absolute quantifications of isolated lipid fractions, (cholesterol and triglyceride), the present invention quantifies the whole lipid moiety (cholesterol, cholesterol esters, triglycerides, and phospholipids), using the fluorescent probe, Nile Red. This fluorescent probe dissolves in all these lipid fractions and can thus document their presence and quantity.
There is a hypothesis today that triglycerides and cholesterol are both involved in arterial wall damage. My approach of global quantification of lipid moiety is able to simultaneously study these two critical fractions. The technique presented is also more simple than those presently in use, and avoids all the complicated steps of lipid fraction quantification and therefore avoids errors. As a consequence, the technique is more precise and it offers more meaningful information as well.
In the future, as more specific markers for each individual fraction are found or synthesized, they can be used to study these specific lipid fractions in an intact lipoprotein particle using the same technique.
Another criticism of current methodology is that lipid determination and apolipoprotein studies are done almost exclusively on fasting samples. This practice ignores the dynamic process of lipid metabolism that occurs after a meal, and in which the consequences of metabolic defects in lipolysis and receptor uptake occur.
Studying fasting samples for lipid moiety or apolipoprotein component offers limited information, and mainly only about the endogenous pathway, reflecting hepatic VLDL synthesis. Humans are not in a continuous fast. They eat at least three times a day; therefore, our lipoproteins are most of the time in a postprandial state. As a result of fatty meals, the lipid component of lipoproteins is continually changing, and the direction of this change is meaningful for at least some known physiological processes: lipid absorption, lipid clearance by intravascular processing by LPL and LDL receptor, dependent or independent tissue uptake. Because the lipid moiety of lipoproteins is in a dynamic state, this generates a dynamic expression of surface epitopes on apolipoproteins by masking (nonexpression) or unmasking (expressing) the epitopes by the lipids (Schonfeld, G. et al., J. Clin. Invest. 64:1288-1297 (1979). It is obvious that dynamic evaluation of lipoprotein particles for these two parameters, the lipid moiety and expressed epitopes on apolipoproteins, is highly desirable. This will generate a multitude of information. The lipoprotein particle has the main function of carrying the fat from the intestines and from the liver to the cells and from the cells to the liver. A challenge with a fatty meal will "put the lipoprotein particles to work." A normal human eats every day approximately 1 gram of fat per kilogram body weight which represents approximately 25 to 35 grams of lipids at each meal. If the lipoprotein transport mechanism is loaded with a standard amount of 70 grams of lipids or 1 gram lipid per kilogram in just one single meal (which represents the approximately 24 hour lipid load for a normal person) this will represent a veritable "lipid stress test." A normal profile of this response as well as an abnormal one will soon emerge, indicating normality or pathology (Chisiu, N.C. Rev. Roum. Biochim. 12(1):75-80 (1975) and Luca, N. et al., Rev. Roum. Biochim.15(2):123-128 (1978).
It is therefore an object of the invention to provide procedures for lipoprotein determinations which overcome these limitations and avoid artifactual results.
It is also an object of the invention to provide a test system which permits the identification of defects in the metabolism and transport of lipids by providing data on the dynamics of relationship between lipid moiety and expressed apolipoprotein epitopes in the various lipoprotein species.